Gibberellins (GAs) are a class of essential hormones controlling a variety of growth and development processes during the entire life cycle of plants, including seed germination, apical dominance, leaf expansion, stem elongation, root growth, floral initiation, another development and fruit maturation (Harberd et al., 1998; Ross et al., 1997; Hedden and Phillips, 2000, Kin and Evan, 2003, Sun and Gubler, 2004; Kende and Zeevaart, 1997, del Pozo, et al., 2005). GAs are substituted tetracyclic diterpene carboxylic acids formed over several biosynthetic steps (Hedden and Phillips, 2000). To date, 136 different GAs have been identified in plants, fungi and bacteria (see for example, the World Wide Web site of plant-hormones.info/gibberellins); however, most of these GAs are precursors or degradation products.
The bioactive GAs synthesized by higher plants are GA1, GA3, GA4, and GA7 (Hedden and Phillips, 2000). The GA biosynthetic pathway can be classified into three stages, with three classes of enzymes involved, including terpene cyclases, cytochrome P450 monooxygenases (CYP450s), and 2-oxoglutarate-dependent dioxygenases (2-ODDs, including GA 20-oxidase, GA 7-oxidase, GA 3-oxidase, and GA 2-oxidase) (Olszewski et al., 2002; Graebe, 1987, Hedden and Phillip, 2000, Sakamoto et al., 2004). Mutants defective in GA biosynthesis have been identified in a variety of plant species, with the most prominent phenotypes being reduced internode length and small dark green leaves (Koornneef and van der Veen, 1980). Other phenotypes include prolonged germination dormancy, inhibited root growth, defective flowering, reduced seed production, and male sterility (King and Evans, 2003, Sakamoto et al., 2004, Tanimoto, 2005, Wang and Li, 2005). Normal growth of these mutants can be restored by exogenous application of active GAs.
GA 2-oxidases (GA2oxs) are a class of 2-ODDs (Thomas, et al., 1999; Sakamoto, et al., 2001, Schomburg, et al., 2003, Sakamoto et al., 2004, Lee and Zeevaart, 2005). The class C19 GA2oxs identified in various plant species can hydroxylate the C-2 of active C19-GAs (GA1 and GA4) or C19-GA precursors (GA20 and GA9) to produce biologically inactive GAs (GA8, GA34, GA29, and GA51, respectively) (Sakamoto et al., 2004). Recently, three novel class C20 GA2oxs, including Arabidopsis GA2ox7 and GA2ox8 and spinach GA2ox3, were found to hydroxylate C20-GA precursors (converting GA12 and GA53 to GA110 and GA97, respectively) but not C19-GAs (Schomburg et al., 2003, Lee and Zeevaart, 2005). The 2β-hydroxylation of C20-GA precursors to GA110 and GA97 renders them unable to be converted to active GAs and thus decreases active GA levels. The class C20 GA2oxs contain three unique and conserved amino acid domains that are absent in the class C19 GA2oxs (Lee and Zeevaart, 2005).
The physiological function of GA2oxs has been studied in a variety of plant species. Arabidopsis GA2ox 1 and GA2ox2 were found to be expressed in inflorescences and developing siliques, which is consistent with a role of GA2oxs in reducing GA levels and promoting seed dormancy (Thomas et al., 1999). Further study with the pea slender mutant, where the SLENDER gene encoding a GA2ox had been knocked out, showed that GA level increased during germination, and resultant seedlings were hyperelongated (Martin et al., 1999). More recently, dwarf phenotype was also found to correlate with reduced GA levels in two Arabidopsis mutants in which GA2ox7 and GA2ox8 were activation-tagged, and ectopic overexpression of these two genes in transgenic tobacco led to dwarf phenotype (Schomburg et al., 2003). These studies demonstrated that GA2oxs are responsible for reducing the endogenous level of biologically active GAs in plants. The class C20 GA2oxs, regulating early steps in the GA biosynthesis pathway, have also been shown to control photoperiods in dicots. In long-day (LD) rosette plants, such as spinach, LD-induced stem elongation and flowering are dependent on GA-regulated processes. In short-day (SD) plants, deactivation of GA53 to GA97 prevails, while in LD plants, conversion of GA53 to the bioactive GA20 and GA1 is favored (Lee and Zeevaart, 2005). The functions of four rice GA2oxs have been previously studied (Sakamoto, et al., 2001, 2004; Sakai et al., 2003).
U.S. patent application Ser. No. 12/139,674 describes the identification and characterization of 10 putative GA2ox genes from the sequence analysis of the rice genome. Differential expression of the GA2ox genes was found to correlate with various developmental processes during rice growth, such as flower development, seed germination and tiller growth. The application also describes methods related to GA2ox genes, such as a method of inhibiting stem elongation and promoting tiller growth in a plant by controlling the expression of a GA2ox gene in a plant.
Features such as semidwarfism, higher finding, more biomass, more adventitious roots, stronger stems and enhanced stress tolerance are the most valuable traits in crop breeding, because they result in plants that are more resistant to damages caused by wind and rain (lodging resistant) and biotic and abiotic stresses, and have stable increase of yields. However, it is difficult to create such plant varieties by conventional breeding of the natural genetic variations of crops species. The present invention offers transgenic approaches for obtaining plants with desirable traits that have not been easily obtained with conventional breading methods, i.e., by controlling expression of GA2ox gene in both monocots and dicots. Plants with more adventitious roots, thicker or stronger stems and branches, more leaf numbers or more biomass, and higher stress tolerance, as well as plants with semidwarfism and higher tillering, have been obtained using the present invention.